The NMDA subtypes of glutamate receptor are involved in synaptic plasticity including processes that may underlie learning and memory. Excessive activation of these receptors after ischemia or brain trauma leads to neurodegeneration. NMDA receptors also play a role in seizure activity, in some chronic neurodegenerative diseases, in nociception, and in psychoses. The receptors are targets for neuroprotective, anticonvulsant, and analgesic drugs. An obstacle to the development of clinically useful NMDA antagonists has been the occurrence of side effects, including psychotomimetic effects and impaired motor function, which are particularly prominent with traditional NMDA antagonists. However, the existence of discrete modulatory sites and the identification of multiple subunits and subtypes of NMDA receptors may lead to the development of antagonists with a novel mechanism of action or antagonists to selectively target subpopulations of receptors that are involved in specific physiologic functions or specific pathologies. Novel NMDA antagonists, which are also useful tools for studies of the distribution, function, and physiology of NMDA receptors, include ifenprodil, which selectively blocks a subtype of NMDA receptor. Endogenous modulators of NMDA receptors include polyamines such as spermine, which potentiates receptor activity. The goals of the present proposal are to study the sites and mechanisms of action of ifenprodil and spermine at NMDA receptors. One approach will involve studies of mutant and chimeric receptor subunits and voltage-clamp recording in oocytes. Ifenprodil and spermine are hypothesized to bind to discrete sites within a regulatory domain (R domain) of the NMDA receptor, that may have an overall structure similar to bacterial periplasmic binding proteins. Thus, a second approach will involve biochemical and ligand-binding studies of purified, soluble R domain proteins, including R domain mutants. A third approach will entail structural studies of purified R domain proteins in the absence and presence of spermine and ifenprodil using X-ray crystallography and liquid chromatography/mass spectrometry. Together, these studies will provide information about the structure and modulation of NMDA receptors that will be valuable in understanding the function of these receptors in the nervous system, their relationships to other receptors and ion channels, and the antagonism of these receptors by novel neuroprotective agents.